Early disease detection and therapy

ABSTRACT

A method for early stage pathology detection, location, imaging, evaluation, and treatment of cells and/or extracellular vesicles in the circulation.

This application claims priority to U.S. Ser. No. 62/303,219 filed Mar.3, 2016 and expressly incorporated by reference herein in its entirety.

A method for early detection of pathologies such as cancer or metabolicdisorders by detecting and evaluating metabolic markers and early stagecells in the retinal circulation. The inventive method renders bloodcellular compositions and components indirectly visible and quantifiableby various imaging modalities. This provides means of early diseasedetection, diagnosis, and treatment.

The retinal circulation stems from the central retinal artery, which isthe first branch of the internal carotid artery. The central retinalartery enters the optic nerve 4 mm-5 mm behind the eyeball, and dividesinto four branches at the optic nerve head, further branching tovascularize the four retinal quadrants.

Retinal tissue is transparent. The retina thus permits visualization andprovides a window into the invisible internal circulation. Takingadvantage of retinal transparency thus provides an opportunity forunhindered visualization and examination of, among other components, thecomposition of blood. As one example, cell types and the metabolic stateof cells, among other features, can be determined. More specifically,photoacoustic technology permits examination, at the molecular level, ofcellular analyte type and concentration, e.g., oxygen, carbon dioxide,sugar, etc. As another example, free circulating tumor cells (CTC) andextracellular vesicles (ECV) such as exosomes can be visualized,assessed, quantified, etc.

Malignant tumors have the capability to metastasize at an early stage,even before the tumor has grown to 3 mm-4 mm in diameter, the point atwhich they are visible by standard imaging techniques such as computedtomography, magnetic resonance imaging, etc. Such small tumors are notspace occupying and often do not produce clinical symptoms in patients,even patients having a genetic predisposition to the pathology are oftennot aware of its development. It is also difficult to diagnoserecurrences of a cancer if a tumor has not yet grown to a size that isradiologically visible. In addition, there is often there is no visibleindication of tumor cell activity within a tumor mass after, e.g.,radiation therapy or at the margin of a resected lesion for a long time.Tumor growth is currently but erroneously considered as the only sign oftumor activity, but there are many examples where metastatic lesionssuddenly grow after years while the patient is considered in remission.

The method permits distinction and differentiation between a malignantlesion and a benign lesion. As only one example, the method candifferentiate a malignant but early melanoma of the choroid, thevascular layer of the eye, from a benign choroidal nevus.

In use, a blood sample is obtained from a patient suspected to have apathology. The blood sample is evaluated for signs of tumor activity byanalyzing the sample for the presence of various molecular tumorbiomarkers, proteins, microDNA, microRNA, enzymes, nucleosomes,extracellular vesicles, free floating tumor cells, etc. In oneembodiment, the blood sample is analyzed for the presence of cancer,and/or various disease biomarkers, including cancer biomarkers, asdisclosed in co-pending U.S. Ser. No. 14/976,321 filed Dec. 21, 2015which is expressly incorporated by reference herein in its entirety.

In one embodiment, an antibody to either a specific tumor or anon-specific tumor is generated in vitro or ex vivo using standardtechniques known the art. The antibody may be any type, e.g.,polyclonal, monoclonal, humanized, aptamers, etc. The generatedantibodies are then used to coat nanoparticles including but not limitedto quantum dots (QD) nanoparticles, forming functionalizednanoparticles. The antibody-coated functionalized nanoparticles areinjected into the patient's circulation, where they target theircorresponding tumor cell membrane receptor antigen. Alternatively, theantibody-coated functionalized nanoparticles are injected in thecirculation where they target and bind to not only to the sessile siteof the tumor, but also target and bind to free circulating tumor cellsand their free floating extracellular vesicles (ECV) or exosomes. Theuser, after waiting for about 1 min to about 8 min for targeting andbinding to occur, then uses photoacoustic technology to image the tumorsite and/or circulating cells, as previously described in U.S. Ser. No.14/976,321.

In one embodiment the nanoparticles range in size from about 1 nm to 500nm, preferably between 1 nm-10 nm. In one embodiment, the nanoparticlesrange in size from about 1 nm-8 nm, 8 mm-20 nm, 30 nm-50 nm, or 50nm-900 nm in diameter.

The exact composition of the functionalized nanoparticle, i.e., both thetype of nanoparticle(s), and the type of antibody coating thenanoparticles, can be specific for one or many biomarkers for differenttumors or tumor types or tumor sets. The nanoparticle can be coated withcell penetrating agents, e.g., cell-penetrating peptide (CPP), activatedCPP (ACPP), etc., eliminating a viral carrier that induces an immuneresponse and cannot be administered repeatedly. That is, the inventivemethod desirably is performed in the absence of a viral carrier.

In one embodiment, the nanoparticles are quantum dots, either alone orin combination with other nanoparticles. As known in the art, quantumdots may be organic, inorganic, synthetic, magnetic, paramagnetic,non-magnetic, nano/microbubble, piezoelectric, etc.; the nanoparticlestructure(s) can include a shell, cage, wire, tube, or otherconfiguration, and the shape may be spherical, cylindroid, tube,multi-faceted, etc.; nanoparticle compositions may be gold, silica,iron, iron oxide, zinc, zinc oxide or their composites, cadmium sulfate,quartz and other piezoelectric nanoparticles, lanthanide or otherupconverting nanoparticles, i.e., quantum dot nanoparticles that areexcited with two photons of a near infrared wavelength or infrared lightand emit one photon of higher energy wavelength of blue to red visiblewavelengths, copper, nickel, carbon, or graphene located in the coreand/or the nanoparticle surface. The composition of the nanoparticle,e.g., gold, iron, iron oxide, etc. may be functionalized with antibodyto specific cell membranes and injected into the circulation to adhereto a tumor, e.g. in the eye, skin, mucosa, etc. The nanoparticles may becoated with a radioactive alpha, beta, or gamma emitter molecule to addthe effect of radiation therapy to other therapies such as photodynamictherapy (PDT), thermotherapy, chemotherapy (drug release), etc. In oneembodiment gold or iron nanoparticles are coated as previously describedto seek the desired cells, then low x-ray radiation is applied to thelesion, e.g., neovascular tissue or a tumor such as melanoma, alone orin conjunction with PDT or thermotherapy. In one embodiment goldnanoparticles are coated as previously described and injected into thecirculation to attach to the desired cells, then low dose thermal orx-ray radiation or femtosecond laser pulses to a continuous pulse laserare provided to produce free radicals. The free radicals damage walls ofendothelial cells, causing a photodynamic effect with free radical andsinglet oxygen formation and platelet aggregation and vascularocclusion. This embodiment may be combined with local drug delivery,such antiproliferative agents, anticancer agents, anti-VEGF agents suchas cisplatin that enhances formation of reactive oxygen species. Thisembodiment may be combined with very low dose systemic administration ofmedication.

In one embodiment, the composition of the circulating elements isdifferentiated by size, as rendered visible in the retinal circulationand recorded, as further disclosed, by the adherent nanoparticles. As anexample, ECV range in size from 100 nm-500 nm, and tumor cells to whichnanoparticles adhere typically range in size from 5 microns-20 microns.

In one embodiment, nanoparticles with a specific anti-tumor antibody,upon stimulation, emit light of a specific wavelength that is detectableby fundus photography, angiography, optical coherence tomography (OCT),and/or OCT angiography (OCTA) as subsequently described. This wavelengthof emitted light provides information on the origin of the specificsessile or particulate circulating in the blood or other fluids to whichthe plurality of nanoparticles containing specific tumor antibodies areattached. Each quantum dot nanoparticle, depending on its size andcomposition after light stimulation, produces a different wavelength oflight, which is specific to its size and composition. If thenanoparticle size or composition and the specific antibody coating (e.g.breast cancer) is known prior to administration in the circulation, thequantum dots seek specific membrane antigens to which to adhere. Whenstimulated by a light beam, they emit a wavelength of light with aspecific wavelength (color) that indicates which nanoparticle/antibodyis attached to which cells or cell types that have the specific cellmembrane receptor, thereby revealing the identity of that circulatingcell, exosome, etc. In one embodiment, after stimulation of the quantumdot nanoparticles with various wavelengths of external light, afluorescein angiography fundus camera records the light emitted bynanoparticles from inside the retinal vessels. These cameras aremodified to have specific barrier filters that separate the incomingstimulating light from the quantum dot emitted exiting light. In thisembodiment, the different wavelength of light (colors) emitted by thestimulated quantum dot nanoparticles in the retinal vessels function asan optical spectroscopic system that are recorded, indicating thepresence of a specific circulating tumor cell and/or ECV in thecirculation. This provides a non-invasive method of imaging, and alsoprovides a method of early cancer detection and differentiation based onwavelength differentiation, i.e., optical spectroscopy), which may thenbe treated by a specific therapy.

In one embodiment, after the nanoparticles have been stimulated withlight of different wavelengths, the nanoparticles emit a fluorescentwavelength that is diagnostic for an actively dividing tumor cell. Thisis based on the principle that the stimulating wavelength of light isdifferent from emitted light from the stimulated quantum dots. Thiswavelength differentiates the nanoparticles by size; a smaller sizeproduces a blue wavelength, whereas a larger size produces a longerwavelength such as red. Because the nanoparticles are functionalizedwith one or more specific antibody or antibodies, it thus alsodifferentiates the nanoparticles bound to a cell containing the specificcell membrane antigen to which the nanoparticles are conjugated. Thus,once the nanoparticles are attached to these circulating cells or ECV,the nanoparticles act as a diagnostic for the presence of the tumor fromwhich the cells or the ECV originate, and they can be tracked andcounted by the camera's photometer. In one embodiment, performing themethod using a plurality of nanoparticles with different sizes and/orcompositions and/or different antibody coatings provides a myriad ofdiagnostic capabilities for diagnosing numerous active tumors, theircellular origin, and their ECVs.

In one embodiment, the retina is radiated, e.g., with focusedelectromagnetic radiation, or low dose microwave radiation, orradiofrequency radiation, to increase the temperature of the circulatingnanoparticles in the retinal and choroidal circulation. Usingphotoacoustic imaging technology, a photoacoustic spectroscopy of theantibody-coated nanoparticles, attached to the free floating cells orexosomes, is obtained. The free floating cells or exosomes are thusimaged, and the specific lesions are diagnosed in the eye or other bodysites, and/or quantified.

In one embodiment, a light beam from a laser is focused continuously ona specific retinal vessel through a contact lens positioned on thecornea of the patient, after application of a topical anesthesia. Theultrasound receiver of the photoacoustic technology system is attachedto any of the side of the eye, lid of the eye, or the forehead proximalto the eye. The operator observes the laser beam focused on a retinalvessel while the retina is under observation through a slit lampmicroscope. The photoacoustic system is connected to the laser deliverysystem via a computer. The operator adjusts the photoacoustic system sothat it controls, via the computer, the thermal energy delivered by thelaser to achieve a photoacoustic or thermoacoustic response, i.e., soundwave, representing a temperature of about 38° C. to 45° C. from theheated nanoparticles. Because the nanoparticles that are circulating inthe retinal vessel absorb the laser energy to a greater degree comparedto their surrounding milieu or environment, the nanoparticles' thermalexpansion produces a photoacoustic sound wave. This photoacoustic soundwave is recorded by the transducer of a photoacoustic unit, indicatingthe presence of circulating nanoparticle-cell complexes and/ornanoparticle-ECV complexes in the retinal vessel. The larger circulatingcells will have more nanoparticles attached to their cell membrane thanthe ten-times smaller extracellular vesicles, and will appear larger byphotoacoustic imaging. Thus, these two structures are differentiatedbased on their size. The origin of these cells or ECV can bedifferentiated by the antibody coated used prior to administration ofthe nanoparticles functionalized to a specific tumor receptor. Usingdifferent laser wavelengths as previously described, with the laserfocused on the retinal vessel the quantum dot nanoparticles that absorba specific wavelength and emit at a different wavelength depending ontheir size, etc. the fundus camera that has a standard fluorescenceangiography system is modified so that a photometer or a detectorabsorbs that presence of different wavelength pulses emitted from thequantum dots as they pass through the laser spot focused on a retinalvessel. This photometer, as other detectors known in the art of opticalspectroscopy, converts radiant power into an electrical signal that canbe processed, recorded, and displayed. This system thus providesinformation on the number of light pulses generated, with the color orwavelength indicating the number of cells or ECV recorded at a giventime. The emitted color is indicative of the quantum dot that producedit, which in turn indicates its size and the identity of the antibodythat coated that size of quantum dot. Because the quantum dots areantibody coated to specific cell membrane receptors, one can determinethe number of those circulating cells or tumor cells, or their ECV, thatare circulating at a given time.

As previously analyzed, the amplitude of this acoustic response is indirect relationship with the size and the composition of thesenanoparticles. The smaller nanoparticles absorb energy faster than thelarger nanoparticles and show a higher temperature than the largernanoparticles. Thus, the smaller nanoparticles expand faster, andproduce a photoacoustic sound with higher amplitude and frequency, thanlarger nanoparticles. Because the nanoparticles are coated with one ormore specific antibody or antibodies against the specific cell ofinterest, e.g. a tumor cell, the origin of the cell type or ECV can berecognized by the photoacoustic unit's processor that differentiates theamplitude and frequency of these sound waves, as in photoacousticspectroscopy techniques, and separates each group of sounds from eachother group of sounds, indicating the number and size of allspecifically coated nanoparticles that have passed through the laserspot light.

As an example, functionalized ferric oxide nanoparticles of a certainsize can be coated with a specific antibody to a specific tumor,indicating the presence of that specific tumor or other tumors in thebody. The groups of nanoparticles, depending upon size, are coated witha different antibody, e.g., those 1 nm-up to 15 nm in diameter arecoated with one antibody, and those 15 nm-up to 30 nm in diameter arecoated with a second antibody, and those 30 nm-up to 50 nm, 50 nm-up to100 nm in diameter, 100 nm-up to 200 nm in diameter, 200 nm-up to 300 nmin diameter, 300 nm-up to 400 nm in diameter, 400 nm-up to 500 nm indiameter, up to about 900 nm, etc., are coated with different antibodiesfor different tumors.

Photoacoustic spectroscopy can quantify and differentiate which tumorshave produced an ECV or circulating tumor cell. Results obtained byphotoacoustic spectroscopy are compared and validated with results usingoptical spectroscopy of the cells using quantum dot nanoparticles thatemit specific wavelengths of light upon stimulation. Using theseresults, one can search for the origin of the tumor using a hand-heldphotoacoustic unit, as described in detail in U.S. Ser. No. 14/976,321filed Dec. 21, 2015 which is expressly incorporated by reference hereinin its entirety.

The origin of these cells or the ECV can be differentiated by theantibody coating used prior to the administration of the functionalizednanoparticles to specific tumor cell receptors. Using different laserwavelength as described above, with the laser focused on the retinalvessel, the nanoparticle that absorbs the specific wavelength and emitsanother wavelength depending on its size etc., the fundus camera thathas a standard fluorescence angiography system is modified so that aphotometer absorbs that presence of different wavelength pulses emittedfrom the nanoparticles as they pass through the laser spot focused on aretinal vessel. This photometer recording acts like an ordinaryspectroscope giving information on the number of light pulses generatedwith what color or wavelength indicating the number of cells or ECVrecorded at a given time. The emitted color is indicative of whatquantum dot produced it, which in turn indicates its size and whichantibody that coated that size quantum dot. Because the quantum dots areantibody coated to specific cell membrane receptors, one can deduct howmany of those circulating cells or tumor cells or their ECV circulate ata given time.

The thermal energy, e.g., electromagnetic radiation, focused microwave,radiofrequency, focused ultrasound wave, etc.) is applied and controlledby the photoacoustic system to any lesion to which the antibody-coatednanoparticles, by specific binding of the antibody to that antibody'sspecific antigen, directs the nanoparticles and to which thenanoparticles. The lesion may be, e.g., in the eye, skin, or otherorgans, etc. Using the photoacoustic system, the site of the lesion isheated to about 38° C. to 42° C. The photoacoustic system produces athermal image of the nanoparticles that are attached, via specificantigen-antibody binding, to the specific tumor mass or infected lesionat any site to which the antibody is directed, in e.g., retina, choroid,skin, mucosa, brain, lung, heart, kidney, liver, temporal artery, etc.Through software within the photoacoustic system, a photoacousticspectroscopy of each the plurality of nanoparticles that are coated withdifferent tumor biomarkers, infective agent, etc. biomarker antibodiescan be created, thereby revealing through their specific and knownantibody coating the nature of the mass, suspected lesion area, infectedtissue, etc. The result thus provides information on the nature ororigin of the tumor cells, infective disease, etc.

In one embodiment, nanoparticles of 1 nm-8 nm can be conjugated with anantibody that, after injection into the circulation of eye cavity,target to specific cell receptors, e.g., receptors on leaky new vessels.The inventive method also provides two- or three-dimensional images byOCTA on the delicate small leaking sites of the blood occular barrier inthe eye, or spinal cord, or brain, etc. due to the presence of thenanoparticle passing through the broken vessel or new vessel.

In one embodiment, antibodies coating, with cell penetrating agents suchas CPP and/or ACPP, etc., inhibitory gene(s) alone or with the CRISPRcomplex, the nanoparticles are directed to microorganisms, e.g.,bacteria, fungi, viruses, parasites, etc. These antibody-coatednanoparticles are injected systemically into the patient. Using thepreviously described photoacoustic technology, the nanoparticles bind tothe corresponding antigens that are present on the free circulatingorganisms, or may be collected in the tissue of the retina, choroid, orelsewhere, e.g., in the skin. If bacterial antibody-coated nanoparticlesare injected into the circulation and accumulate in the eye or skin, thenanoparticles can be heated by the previously described specific laserwavelength, producing a photoacoustic response that can be recorded by aphotoacoustic system. This embodiment provides information about theorigin of the infection, etc.

Nanoparticle assisted photography, angiography, OCT, OCT angiography(OCTA) is used for detection and therapy of a disease process, e.g., inthe eye or elsewhere in the body.

OCT is based on low coherence tomography using light, and capturestwo-dimensional or three-dimensional images from the light scatteredthrough and in the media through which it propagates. Usingophthalmology as an example because of transparent ocular media, OCTprovides information about the anterior segment, the lens and theretina/anterior choroid. OCT has a potential application for imagingskin and its various disorders, and provides information on thethickness and borders of a skin lesion.

OCTA technology can image the retinal/choroidal vessels, vascularabnormalities, and any retinal/choroidal neovascularizations. In OCTA,consecutive B-scan images of the laser light provide a two- orthree-dimensional cross-sectional image of tissues, such as the retina,to the limit of light penetration in the tissue. OCTA is based on thealgorithm of split-spectrum amplitude decorrelation, which allowsvisualization of the inner and outer layer of the retinal vasculatureand the choriocapillary of both inner and outer retinal vascular plexusand the choroidal capillary layer. OCTA contrasts the images obtainedfrom the static tissue, e.g. retinal tissue, vs non-static tissue suchas blood vessels. OCTA decorrelates the signals, using associatedsoftware, and presents a three-dimensional image of the retinal vesselsand choriocapilaries separately to a depth of about 10 micron below theBruch's membrane.

Currently available OCTA systems provide an average axial resolution ofabout 15 micrometer and an average lateral resolution of about 5microns. However, the resolution of the image is not sufficient toprovide a signal from small molecules having a diameter of less than 500nm-1 micron. Therefore at present, suspensions of, e.g. serum molecules,originating from leaky vessels in the choroid and retina, remaininvisible with OCTA and appear as a dark space in OCT images. Usingfluorescein angiography (FA), a much older technique, one sees passageof a fluorescein dye through the small discontinuity in the damaged wallof a leaky vessels. In a fluorescein angiography (FA) the dye becomesvisible as a stained wall of a damaged vessel, or as a pool of dyecollected in the interstitial spaces or cavities of diseased tissue,e.g. retina. Using fluorescein, the collected small molecules of thefluorescein dye remains invisible, or appear as dark spaces in the OCTand OCTA images and/or under the sensory retina. Therefore, OCT or OCTAdo not compare well with standard fluorescein angiography indemonstrating leakage from abnormal or neovascular lesions or smalldefects in the retinal pigment epithelium or abnormal retinal vessels.The abnormal vessels or new vessels result from hypoxia causing adjacenttissues to produce vascular endothelial growth factor (VEGF), inducingneovascularization. Inflammatory diseases directly affect the bloodretinal barrier, cause leakage of fluorescein outside the retinalvasculature. Pathologies such as age-related macular degeneration(ARMD), diabetic retinopathy, central serous retinopathy, stroke, etc.all induce damage to the retinal vasculature. In one embodiment,nanoparticles carry antibodies to VEGF to inhibit growth-inducingabnormal vessels or the vascular supply of a tumor.

FA has a number of shortcomings. One shortcoming is that the deeperretinal capillary plexus and radial peripapillary capillary networkcannot be seen in FA due to scattering of the incidence light (blue) andreflected fluorescent light, and in the deeper retinal layers.Similarly, the invisibility of the peripapillary network in the FArenders FA not useful for evaluation of the progression of damage to theoptic nerve head or retina in patients with glaucoma, etc. FA also haspotential side effects such as nausea, vomiting, pruritus, urticaria,pyrexia, thrombophlebitis, and life threatening reactions such asanaphylaxis response, bronchospasm, cardiac arrest, and death. Localtissue necrosis can also occur with extravasation of the dye at the siteof the injection. In addition the fluorescin angiogram cannotsatisfactorily demonstrate the choroidal vessels well. As a result, thepatient must undergo a second minimally invasive procedure, i.e., theso-called indocyanin green angiography, in which an infrared light isused to excite the dye, obtaining a fluorescent infrared wave length forimaging. Indocyanine green angiography has equal if not more sideeffects than fluorescein angiography, particularly life threatening sideeffects or death in patients with seafood or iodine allergies. Anotherproblem is that both these dyes clear the circulation within a fewminutes, and have no specificity to abnormal endothelial cells. They arealso invisible with OCT technology because of their small molecularsize. They also have no therapeutic effect on the disease process andare used only as an imaging tool.

The inventive method overcomes these deficiencies. It provides a methodof angiography with new fluorescent quantum dot nanostructures. Thequantum dots are themselves fluorescent; when stimulated by a specificwavelength of light, they emit a longer wavelength of light (fluorescentlight). They act like fluorescein molecules, but are larger in size andtheir fluorescence does not bleach. In contrast, in fluoresceinmolecules, fluorescence bleaches and turns itself off beyond a certaintime of light exposure. The quantum dots of 1 nm-8 nm can pass throughthe blood brain barrier at a site of minor discontinuity. As previouslydescribed, the cause of the discontinuity may an inflammatory process,abnormal vessels caused by hypoxia, vascular occlusion such as indiabetes or stroke, a neoplastic lesion, an infection, trauma, tumor,etc.

In one embodiment, quantum dot nanoparticles fluoresce, upon exposure toa specific wavelength of laser or diode light, at a specific wavelengthwith lower energy. Fluorescence or emitted light from the quantum dotnanoparticles passes, without being scattered by the transparent retinaltissue, through an appropriate barrier filter and can be captured byphotograph or video camera.

In one embodiment, the light source that excites the nanoparticles, suchas an infrared (IR) source, can simultaneously produce an OCT or an OCTAand which can be recorded separately by an OCT unit and software toproduce OCT or OCTA images. The light that is generated from the quantumdot nanoparticles can also be separated from that of the OCT unit via anappropriate barrier filter diverted to be imaged by an angiographiccamera separately, creating a combined OCT, OCTA, and fluorescentangiogram system. In one embodiment, infrared excitation light andlanthanide group nanoparticles are used because of the advantage of notbeing seen by the patient's photoreceptors, thus it does not blind thepatient as does visible light. In addition, the same light cansimultaneously produce the OCT and OCTA images. This unit requires onlya simple infrared prism to divert the returning infrared light to theOCT unit, while permitting the emitted visible light coming through fromthe lanthanide nanoparticles to be picked up by the system's camera.

In one embodiment, the system provides an IR wavelength excitation andnear-IR emission for use with nanoparticles of 1 nm up to 150 nm or evenlarger than 150 nm. The following elements or composites upconvert thelight by absorbing two photons of a low energy light beam and upconvertit to one photon of higher energy beam, e.g., infrared to yellow or bluewavelengths, etc.: lanthanide nanoparticles family erbium, thulium,holium Ln, Ti, Ni, Mo, Re, Os, cerium, lanthnum, lutetium, yttrium,scandium, gadolinium fluoride, and lanthnum fluoride, or as a lanthaniderich composite as known in the art having one lanthanide ion from thegroup of Ce, Nd, ER, etc. in combination with an anion from a halide,phosphate, vanadate etc., or LaAL0ri3/SrTiO3, thulium-doped silica,gadelonium or neodynium complexes such as Nd—Fe—B, or semiconductorsCdSe, PbSe, PbS. These nanoparticles upconvert the excitation light,converting two IR photons to a single visible light that can bephotographed with a standard fundus camera, or video-angiography can beperformed without producing the previously described side effects offluoresceine or indocyanin green angiography.

In one embodiment, these nanoparticles are coated. The coating may bepolymers such as (poly)ethylene glycol (PEG), chitosan, cell penetratingagents such as CPP, ACPP, etc., one part of the binding pairbiotin/streptavadin, etc., as well as containing specific ornon-specific antibodies to the desired cells. The antibodies seekabnormal vessels, e.g., found in age related macular degeneration or atumor, and adhere to the vessel walls for subsequent treatment, under anoperator's control and observation. The nanoparticles in any of theseembodiments are injected in a biocompatible formulation for the variousdescribed indications. In one embodiment, coated nanoparticles arefunctionalized by conjugation with various cytokine antibodies. Thesecytokine antibodies, when injected into the circulation, attach tocytokine producing cells, e.g., anti-VEGF coated nanoparticles willlocalized in an area of a retina that is ischemic and thus that producesVEGF, IL-1, etc. The nanoparticles may be conjugated with cellpenetrating peptides such as CPP or ACPP, etc. and an inhibitorygene/CRISRP cas9, to inhibit production of VEGF, other genes, siRNA,etc. or conjugated with IL inhibitory medication. Many diseases such ascancer produce an inflammatory environment, and diseases such asAlzheimer's disease or traumatic brain injury produce inflammatoryinsults. The inventive technology not only demonstrates and images thesite of inflammation, but also potentially treats the inflammation withappropriate medication administered with the quantum dot nanoparticles.The system can also be used for an early stage disease process that canbe treated, and the results of the treatment can be quantified.

This methodology can demonstrate the presence of the inflammatorybiomarkers in some areas, indicating a part of a tissue affected by apathology such as an infarct, or a disease state such as aneurodegenerative disease, e.g., Alzheimer's disease, infection, anautoimmune response, a genetic disorder, a tumor, etc. This area can beimaged photographically or using X-rays, MRI, CT, PET, etc., or byantibody coated nanoparticle assisted photoacoustic imaging or localizedthermotherapy using electromagnetic radiation, RF, microwaves, focusedultrasound, alternating magnetic field, etc., and/or simultaneous drugrelease from the nanoparticles. In one embodiment, the nanoparticles,e.g., a lanthanide etc., is coated with a thermosensitive polymer suchas chitosan and conjugated with an anti-VEGF cell specific antibody thatattaches to vascular endothelial cells producing VEGF receptors in theretina or choroidal abnormal vessels or elsewhere in tumor vasculature.One can specifically make these abnormal vessels visible, image them,and treat them with various means such as a laser in the same session orby another source of energy such as microwave, radiofrequency (RF)waves, electromagnetic radiation, or focused ultrasound waves heatingthe nanoparticles.

In one embodiment, the IR light can also be simultaneously used as inOCT technology to provide an OCTA image of the retina, or as ananoparticle-assisted angiogram. The angiogram images can besuperimposed, by software, on the OCTA or the OCT to show the exactlocation of the nanoparticle, e.g., in vessels, or in cysts or cavities,or in the subretinal fluid produced by various diseases of the retinaand the choroid. In one embodiment, nanoparticles with a size rangingfrom 1 nm-8 nm can pass through the smallest breaks of the retinalvessels or retinal pigment epithelium, and be visualized duringnanoparticle assisted angiography. This beneficially combines twosystems, angiography and OCTA, in one instrument.

In one embodiment, nanoparticles ranging in size from 1 nm-8 nm, or from8 nm-900 nm generate an internal light after excitation and emit visiblelight. In this embodiment, the emitted wavelength provides betterinformation from deeper structures, or from the optic nerve head, or inthe posterior choroidal circulation, with emission wavelengths appearingakin to stars shining in a dark sky. This emission wavelength can berecorded as a photograph or an angiogram. Because this emitted light isgenerated internal to a tissue and is not reflected, it does not createinterference with incoming invisible IR light. It thus can be seen moredistinctly than is possible by standard fluorescein angiography. Byselection of the nanoparticle composition, such as quantum dot/plasmonicnanoparticles, one can image the deeper structures located in thechoroidal circulation than previously possible with simple OCTA. Forexample, using nanoparticles from one of the coated functionalizedlanthanide series, which then emit visible light and can be visualizedand recorded, IR light penetrates deep in the choroid and excites thosenanoparticles, which can be visualized and recorded. In one embodiment,the nanoparticles are subjected to wavelength from an IR laser to bothheat the nanoparticles and release any medication from thethermosensitive coating, such as chitosan, of a nanoparticle, e.g.,anti-VEGF, anticancer drugs, antibiotics, anti-inflammatoryagents/inhibitory genes, etc. The wavelength also heats abnormalendothelial cells to enhance the effect of anti-VEGF on the abnormalvessels.

In one embodiment, a known photosensitizer, e.g., verteporphin, isconjugated with antibody coated nanoparticles having a thermosensitivepolymeric coating such as chitosan. Upon light activation, thenanoparticles simultaneously release a small amount of photosensitizerfrom the polymeric coating. Subsequently, after release of thephotosensitizer, in the presence of oxygen and laser application,singlet oxygen and free radicals are created. This damages the wall ofthe abnormal vessels, causing platelet aggregation which closes abnormalvessels, beneficially without damaging normal adjacent tissue orcreating a systemic response to the photosensitizer. The inventivemethod thus is an improvement over and is in contrast to originalphotodynamic therapy that produced side effects after intravenousinjection of a large amount of photosensitizer, such as skin burns whenthe patient was exposed to sunlight, or an allergic response even assevere as shock or death, or tissue necrosis with photosensitizerextravasation. Nanoparticles produce a fluorescent light upon laserirradiation. This can be simultaneously imaged while the desired tissueis treated and the borders of the neovascular tissue or the lesionbecomes visible by the attached antibody coated nanoparticles. It is ofnote that standard photodynamic therapy for abnormal vessels in ARMD orfor neoplasms does not provide any information on the borders of thetumor during therapy. Thus, the investigator has to treat the lesionfrom the memory and not objectively. This led to many recurrencesrequiring treatment that unnecessarily damaged the overlaying healthyretina, among other drawbacks. The inventive method solves this problemby allowing visualization of tumor borders and simultaneous release ofmedication.

In one embodiment, passage of the sized nanoparticles from thecirculation provides information on the location and the degree of thedamage caused by the disease. Lesions as small as one nm or as large as900 nm can be imaged. For example, if a damaged area of a vessel exceeds5 microns, erythrocytes will exit the vessels and cause frank bleedingthat obscures the bleeding site. Lesions smaller than 2 microns wouldallow only non-cellular fluid, i.e., serum, to escape, so this would notbe visible by standard OCTA. In contrast, functionalized quantum dotnanoparticles can pass through nanosized openings in the vessels, remainattached to damaged cells, and can be visualized for several hours ordays at the site. This permits repeat quantum dot assisted OCTA orangiography over many days, following the lesion during a post-operativeperiod non-invasively using the inventive system.

In one embodiment, since the absorption wavelength of the nanoparticlecan be matched with the wavelength generated by OCT, it is possible toexcite the nanoparticles to fluoresce. The wavelength of the fluorescentlight can be matched with the absorption wavelength of thephotosensitizer, e.g. amino-levulinic acid etc., to induce singletoxygen and free radical formation in a tumor, inducing endothelial cellwall damage of the lesion as many times as needed for therapy. If vesselleakage has persisted and requires re-sealing, this can be accomplishedby application of minor thermal laser energy that is absorbed by thenanoparticle and that heats the surrounding area. This is helpful intreating the wet form of age-related macular degeneration, one of themajor cause of blindness due to abnormal vessel growth and bleedingunder the retina, or diabetic retinopathy with macular edema havingearly or late stage proliferative retinopathy. Alternatively, in thecase of an invisible bleeding site, low level X-ray application can bedone. In the presence of an antibody-coated nanoparticle of gold or agold composite, the functionalized gold nanoparticles enhance the localeffect of the radiation while protecting other areas. The sametechnology can be applied to localized cancers, e.g., breast, prostate,brain, lung, etc. One can also easily repeat treatment, if needed, witha radioactive coating of the antibody-coated quantum dot nanoparticlesin cancer patients.

In another embodiment, the nanoparticles may be conjugated with cellpenetrating agents such as CPP, ACPP, etc. to a gene/CRISPR cas9 complexthat can modify the genetic composition of the cells to which thenanoparticles are targeted. Such a cell may have a genetic abnormality,e.g., age-related macular degeneration etc. and, in this embodiment, thefunctionalized nanoparticles/genes along with CRISPR cas9 are picked upby the targeted cells, and release the gene into the cell upon or aftercellular uptake. As one example, a gene to correct the geneticabnormality in retinitis pigmentosa can allow retinal cells to regainsensitivity to light. As another example, a first conjugated gene isfrom the opsin family, and a second conjugated gene/CRISPR cas9 complexmodifies the genetic composition of cells that might have a geneticabnormality such as retinitis pigmentosa, epilepsy, Alzheimer's disease,Parkinson's disease, etc. As another example, the conjugated gene is anopsin family member such as rhodopsin, halorhodopsin, etc. In anotherembodiment, the antibody-coated nanoparticle containing the conjugatedgene/CRISPR cas9 complex to eliminate the tumor producing gene, orhaving an inhibitory gene, siRNA, etc. is injected intravenously in aneye or elsewhere as needed. In another embodiment, theantibody-nanoparticle is directed to retinal tumor or brain tumor cells,e.g., retinoblastoma or glioma or glioblastoms. After release of therhodopsin gene/CRISPR cas9 complex from a thermosensitive coating, onecan convert these non-excitable cells to light-sensitive tumor cellsthat response to light by being depolarized. One can applynon-coagulative pulsed laser fora period of time of about 10 min-15 minor more to the tumor. By applying repeated continuous rapid laser pulsesunder control of a photoacoustic system, as previously described, usingan externally located laser or by a fiber optic, the cell membrane ofthe tumor cells depolarize. As a result, the tumor is rendered permeableto anticancer or other medication that is released from the coating ofthe nanoparticle, freely diffusable in the tumor cells to damage thetumor cells.

The tumor can be of neuronal origin such as a glioblastoma in the brainor a retinoblastoma in the eye, or an acoustic neuroma or meningioma, orthe tumor may be from the spinal cord cells or peripheral nerves. Thetumor can be a melanoma of the choroid or a skin melanoma, or the tumorcan be of mesenchymal cells or of ectodermal origin. This techniquerepresents a completely new approach of eliminating the tumor cellswithout burning them or removing them surgically. By choosingfunctionalized piezoelectric nanoparticles, one can also use ultrasoundor focused ultrasound for both cell depolarization and thermotherapy asneeded. Because ultrasound travels deep in tissues, it can be usedadvantageously in deep body or brain tissues. Nanoparticles are coatedwith a biocompatible coating such as PEG, CPP, ACPP, etc. orthermosensitive polymers such as chitosan that dissolve during lasertherapy and releases a medication, e.g. an anti-VEGF antibody and/or anyother medication e.g. anticancer agents melphalan etc. useful to treat atumor such as a retinoblastoma, glioblastoma, medulloblastoma, acousticneuroma, spinal cord tumor, peripheral nerve tumor, choroidal melanoma,skin melanoma, mesenchymal cell tumor, tumor of ectodermal origin, basalcell carcinoma, squamous cancer, etc., or an anti-melanoma medicationsuch as allovectin-7, decarbazine, or others such as alkylating agentssuch as nitrogen mustards including mechlorethamine, cyclophosphamide,melphalan, chlorambucil, ifosfamide and busulfan; nitrosoureas includingN-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) andsemustine (MeCCNU), fotemustine and streptozotocin; tetrazines includingdacarbazine, mitozolomide and temozolomide; aziridines includingthiotepa, mytomycin and diaziquone (AZQ); cisplatin and derivativesincluding cisplatin, carboplatin, and oxaliplatin; and non-classicalalkylating agents including procarbazine and hexamethylmelamine;anti-metabolites such as anti-folates including methotrexate andpemetrexed; fluoropyrimidines including fluorouracil and capecitabine;deoxynucleoside analogues including cytarabine, gemcitabine, decitabine,Vidaza, fludarabine, nelarabine, cladribine, clofarabine andpentostatin; and thiopurines including thioguanine and mercaptopurine;anti-microtubule agents such as vinca alkaloids including vincristineand vinblastine, semi-synthetic vinca alkaloids include vinorelbine,vindesine, and vinflunine; taxanes including paclitaxel and docetaxel;topoisomerase inhibitors including irinotecan, topotecan, etoposide,doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, andaclarubicin; cytotoxic antibiotics including actinomycin, bleomycin,plicamycin, mitomycin, doxorubicin, daunorubicin, epirubicin,idarubicin, pirarubicin, aclarubicin, and mitoxantrone, etc. to treatmelanoma of the choroid or a skin lesion, such as basal cell carcinomaor squamous carcinoma of the skin, etc.

In one embodiment, the antibody coated nanoparticles are coated with animmune checkpoint inhibitor to facilitate leukocyte mediated tumordestruction. Immune checkpoint inhibitors include, but are not limitedto, monoclonal antibodies that target either PD-1 or PD-L1 includingpembrolizumab (KEYTRUDA®) and nivolumab (OPDIVO®), monoclonal antibodiesthat target CTLA-4 such as ipilimumab (YERVOY®), and monoclonalantibodies directed against at least one of CD27, CD28, CD40, CD122,CD137, OX40 (also termed CD134), GITR (glucocorticoid-induced TNFRfamily related gene), ICOS (inducible T-cell costimulatory also termedCD278), A2AR (adenosine A2A receptor), B7-H3 (also termed CD276), B7-H4(also termed VTCN1), BTLA (B and T lymphocyte attenuator also termedCD272), CTLA-4 (cytotoxic T-lymphocyte-associated protein 4 also termedCD152), IDO (indoleamine 2,3-dioxygenase), KIR (killer-cellimmunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), PD-1(programmed death 1), PD-L1 (programmed death ligand 1), TIM-3 (T-cellimmunoglobulin domain and mucin domain 3), VISTA (V-domain Ig suppressorof T cell activation), and other to be developed.

In one embodiment, the nanoparticles are heated by the same laser thatis used to create a fluorescent response, or by another laser producinga wavelength that is absorbed by the nanoparticles to heat thempreferentially over the surrounding tissue, e.g., retina or choroid. Thenanoparticle temperature is heated to about 43° C., which is below thethermal pain threshold sensation. This heats and thus damages tumorcells by the method previously described in U.S. Pat. No. 8,210,184which is expressly incorporated by reference herein in its entirety.

In one embodiment, thermotherapy is combined with immune therapy byincluding the targeted nanoparticles conjugated with immune stimulators,such as modified viral particles or modified plant viral viruses, alongwith the nanoparticles etc. to induce an immune response to the tumorcells, disease process, Alzheimer's plaques, etc.

In one embodiment, imaging is combined with nanoparticle assistedphotoacoustic imaging, OCT, OCTA, FA, focused ultrasound, non-focusedultrasound, MRI, PET scan, CT scan, surface enhanced Raman spectroscopyand imaging, which enhances the molecular diagnosis of a substanceattached to the surface of a metallic nanoparticle after it is exposedto the laser light energy as known in the art, etc. recorded to enhancean early disease diagnosis in vivo.

In one embodiment, one can deliver antibiotics, anti-parasitic drugs,anti-fungal drugs, anti-viral drugs anti-inflammatory agents, etc.conjugated with the quantum dot nanoparticles.

The embodiments shown and described in the specification are onlyspecific embodiments of inventors who are skilled in the art and are notlimiting in any way to the location in the body, nanoparticle type orcomposition, disease process, source of energy, medication, gene, etc.Therefore, various changes, modifications, or alterations to thoseembodiments may be made without departing from the spirit of theinvention in the scope of the following claims. The references cited areexpressly incorporated by reference herein in their entirety.

What is claimed is:
 1. A method for early stage detection and treatmentof a pathology in a patient, the method comprising the following steps:administering a pathology-specific antibody-coated nanoparticle into apatient's circulation, the pathology-specific antibody-coatednanoparticle comprising a nanoparticle bound to a pathology-specificantibody and further conjugated with an immune checkpoint inhibitor inthe form of a monoclonal antibody, wherein the pathology-specificantibody and monoclonal antibody are different, allowing a time intervalfor the pathology-specific antibody of the antibody-coated nanoparticleto attach to a specific antigen in a circulating vesicle or cell,thereafter exciting the antibody-coated nanoparticle with an energysource in a retinal vessel in the patient to elicit ananoparticle-specific wavelength of light response from theantibody-coated nanoparticle attached to the circulating vesicle or cellindicating an early stage pathology in the patient due to the presenceof the specific antigen to which the pathology-specific antibody of theantibody-coated nanoparticle is bound, imaging and quantifying thenanoparticle-specific wavelength of light response in the retinal vesselin the patient for detection of the early stage pathology prior to amass associated with the early stage pathology growing beyond a diameterof 3-4 mm and causing a clinical symptom, and treating the pathologyusing the monoclonal antibody so as to facilitate leukocyte mediateddestruction, wherein the pathology is cancer.
 2. The method of claim 1where the circulating vesicle or cell originates from a malignant tumor,the method further comprising diagnosing the malignant tumor prior tothe malignant tumor having reached a size up to 3 mm in diameter.
 3. Themethod of claim 1 where the nanoparticle is a quantum dot theadministering step is by injection; the antigen is at least one of areceptor on a tumor cell membrane, a protein in a blood vessel wall, asessile tumor, a free circulating tumor cell, an ECV, an infectiveagent, or an exosome; and the method further comprises applying thermalenergy to the antibody-coated nanoparticle attached to the specificantigen in the circulating vesicle or cell, and receiving aphotoacoustic sound from the heated antibody-coated nanoparticleattached to the specific antigen in the circulating vesicle or cell toimage the circulating vesicle or cell.
 4. The method of claim 3 wherethe nanoparticle has a size ranging from 1 nm-8 nm, 8 nm-20 nm, 20 nm-50nm, 50 nm-100 nm, 100 nm-500 nm, or 500 nm-900 nm.
 5. The method ofclaim 3 where the imaging and quantifying steps are by fundusphotography, nanoparticle assisted angiography, optical coherencetomography (OCT), OCT angiography (OCTA), and combinations thereof. 6.The method of claim 1 where the pathology is at a site selected from thegroup consisting of retina, choroid, skin, mucosa, and combinationsthereof.
 7. The method of claim 5 where the imaging and quantifyingsteps are by OCTA and at least one of a two-dimensional and a threedimensional image of the antibody-coated nanoparticle is provided. 8.The method of claim 1 where the antibody-coated nanoparticle contains anadditional coating.
 9. The method of claim 8 where the additionalcoating is selected from the group consisting of a polymer, athermosensitive polymer, chitosan, a cell penetrating agent, a part of abiotin/streptavadin binding pair, an antibody, an antigen, andcombinations thereof.
 10. The method of claim 9 where the additionalcoating is a thermosensitive polymer, and a photosensitizer isconjugated to the thermosensitive polymer.
 11. The method of claim 3where the antibody-coated nanoparticle is conjugated to a gene that canmodify the genetic composition of the circulating vesicle or cell towhich the antibody: coated nanoparticle is attached.
 12. The method ofclaim 3 where the antibody-coated nanoparticle further comprises athermosensitive coating and an anticancer agent, and the method furthercomprises applying repeated non-coagulative laser pulses for about 10min-15 min controlled by a photoacoustic system using an externallylocated laser or a fiber optic to increase a temperature of thecirculating vesicle or cell to 42° C. to 43° C. to depolarize thecirculating vesicle or cell, release the anticancer agent to thecirculating vesicle or cell, render the circulating vesicle or cellmembrane permeable to the anticancer agent that is released from thethermosensitive coating of the antibody-coated nanoparticle to freelydiffuse the anti-cancer agent in the circulating vesicle or cell todamage the circulating vesicle or cell.
 13. The method of claim 12 wherethe cancer is a glioblastoma, a retinoblastoma, a medulloblastoma, anacoustic neuroma, a spinal cord tumor, a peripheral nerve tumor, achoroidal melanoma, a skin melanoma, a mesenchymal cell tumor, a tumorof ectodermal origin, a basal cell carcinoma, a squamous cancer, andcombinations thereof.
 14. The method of claim 12 where the anticanceragent is selected from the group consisting of melphalan, decarbazine,5-fluorouracil, mechlorethamine, cyclophosphamide, melphalan,chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU),carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine,streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa,mytomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin,procarbazine, hexamethylmelamine, methotrexate, pemetrexed,fluorouracil, capecitabine, cytarabine, gemcitabine, decitabine,azacitidine, fludarabine, nelarabine, cladribine, clofarabine,pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine,vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, irinotecan,topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin,merbarone, aclarubicin, actinomycin, bleomycin, plicamycin, mitomycin,doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin,aclarubicin, mitoxantrone, and combinations thereof.
 15. The method ofclaim 1 further comprising analyzing a blood sample from the patient fora component selected from the group consisting of a biomarker, a tumorprotein, a microDNA, a microRNA, an enzyme, a nucleosome, anextracellular vesicle, a free floating tumor cell, a biomolecule, andcombinations thereof.
 16. The method of claim 1 where the imaging stepuses a method selected from the group consisting of antibody-coatednanoparticle assisted photoacoustic technology, ultrasound, surfaceenhanced Raman spectroscopy, nanoparticle assisted fluorescenceangiography, photography, computed tomography, magnetic resonanceimaging, and combinations thereof.
 17. The method of claim 1 where theantibody-coated nanoparticle is further coated with an immune stimulatorin the form of modified viral particles or modified plant viruses, andwhere the method further comprises treating the pathology using themodified viral particles or modified plant viruses to induce an immuneresponse to tumor cells.
 18. A recognition and treatment method for anearly stage of a tumor without producing a clinical symptom in apatient, the method comprising: defining a change in a tissue of apatient, the change indicating an early stage lesion in at least onecell in the tissue, administering a plurality of nanoparticles to thepatient, where the nanoparticles are bound to an a lesion-specificantibody against a protein in the lesion and are coated with athermosensitive polymer containing an immune checkpoint inhibitor in theform of a monoclonal antibody for therapy of the lesion, wherein thelesion-specific antibody and monoclonal antibody are different,interrogating the cellular and extracellular vesicles in the patient'sblood using a photoacoustic spectroscopic system and an opticalspectroscopic fundus camera equipped with a laser that is focused on aretinal vessel of the patient, recording, imaging, quantifying, and/oranalyzing the patient's cells and extracellular vesicles according tothe nanoparticles in the retinal vessel in the patient, treating thelesion using the immune checkpoint inhibitor so as to facilitateleukocyte mediated tumor destruction, and using the system to increasethe temperature of the nanoparticles to a temperature of 42° C. to 43°C. to release the monoclonal antibody from the thermosensitive polymerto treat the lesion.
 19. The method of claim 18 further comprisingexposing the nanoparticles to a thermal energy source from a thermalenergy delivery unit controlled by the photoacoustic spectroscopicsystem to increase the temperature in the nanoparticles, resulting in aphotoacoustic sound that is recorded by the photoacoustic spectroscopicsystem, the method combining photoacoustic imaging to verify and imagethe location of the early stage lesion.
 20. The method of claim 19 wherethe photoacoustic spectroscopic system controls the amount of thermalenergy to maintain a system operator desired temperature ranging from38° C. to 45° C.
 21. The method of claim 18 where the nanoparticles areselected from the group consisting of quantum dots, organic, inorganic,synthetic, magnetic, paramagnetic, non-magnetic, nano/microbubble,piezoelectric, shell structure, cage structure, wire structure, tubestructure, spherical, cylindroid, tube, multi-faceted, gold, silica,iron, iron oxide, zinc, zinc oxide or their composites, cadmium sulfate,lanthanide, upconverting nanoparticles, copper core or surface, nickelcore or surface, carbon core or surface, graphene core or surface,radioactive surface, and combinations thereof.
 22. The method of claim18 further comprising stimulating the nanoparticles using a lightsource, and imaging the nanoparticles using optical coherence tomographyangiography (OCTA) so as to determine the location of the lesion.